For each fragment, specific primers with appropriate restriction sites
were designed and used.
Evaluation of a novel method based on amplification of DNA fragments surrounding rare restriction sites
(ADSRRS fingerprinting) for typing strains of vancomycin-resistant Enterococcus faecium.
Primers used for amplification of lipase gene with additional of NdeI and SalI restriction sites
No Primer Nucleotide sequence(5'[right arrow]3') 1 Feksp CAACATATGAACAAGAACAAAACCTTGCTCGCC 33 2 Resksp AAAGTCGACGAGCCCCGCGTTCTT 24 No Primer [SIGMA] basa Tm ([degrees]C) 1 Feksp 61 2 Resksp 60
In order to clone the fragment containing the [beta]-globin gene promoter in a pBluescript II SK (pSK) vector, [beta]_KpnI and [beta]_XhoI primers, engineered to contain both KpnI and XhoI restriction site
, respectively, were used.
Locations of the restriction sites
(black hash marks), bacterial transposon Tn7 sites (grey triangles), polyhedrin promoter (angled arrow corresponding to the sequence below), SINV-3 open reading frames (dark closed arrows), and approximate location of the area detected by the polyclonal antibody preparation (pAb) are shown.
Therefore, longer fragments affected by ADO could be misinterpreted as alleles without a mutation in the restriction site
. This problem can be avoided with ddRAD, for which two restriction enzymes are utilized (Davey et al., 2013).
Oligonucleotides used to amplify CLCuKoV promoter were P1 (5' GTTGACTAAAATTGAATCACC-3') as forward with the addition of SacI restriction site
and P2 (5'- CAAACGCATACTTAGCAACG-3') as reverse primer with the addition of SalI restriction site
Faloona et al., "Enzymatic amplification of [beta]-globin genomic sequences and restriction site
analysis for diagnosis of sickle cell anemia," Science, vol.
(5) Restriction sites
introduced to forward and reverse primers for amplifying VH are chosen based on restriction sites
of pFUSEss-CHIg-hG1 in the 5' to 3' direction, including EcoRI, EcoRV, XhoI, and NheI.
The RsaI peak at 400 bp matched the restriction site
in several [gamma]-proteobacteria, including that of 37 Vibrio species, as well as several more species from Firmicutes and [alpha]-proteobacteria.
The purified 186-bp DNA fragment corresponding to the first huIFN[alpha]2 synthetic fragment was cloned into pGX4T1/rhuIFN[alpha]2a (wild type cDNA sequence) previously cut at the 5' EcoRI and 3' BglII restriction sites
to generate a plasmid containing only the 48 bp corresponding to the 16 residues at the huIFN[alpha]2 COOH terminus.
For the alignment of A DNA digest, the first 2 cycles on both ends were omitted because they corresponded to the restriction site
sequences and because the domination of certain bases in the first cycle caused calibration problems in the image analysis software.